Wensel T G, Stryer L
Department of Cell Biology, Sherman Fairchild Center, Stanford University School of Medicine, California 94305.
Proteins. 1986 Sep;1(1):90-9. doi: 10.1002/prot.340010114.
The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.
在视网膜视杆细胞外段，活化的转导素（Tα-GTP）开启环磷酸鸟苷磷酸二酯酶（PDE）是视觉兴奋过程中的关键步骤。胰蛋白酶通过降解PDE（αβγ）的γ亚基来激活该酶，而γ亚基又能逆转这种激活作用，据此有人提出Tα-GTP通过解除γ亚基施加的抑制性限制来激活PDE（赫尔利和斯特里尔：《生物化学杂志》257:11094 - 11099，1982年）。我们在此报告的研究表明，添加γ亚基也能逆转Tα-GTP-γS对PDE的激活作用。本文还介绍了一种高产率（50 - 80%）制备γ亚基的方法。十二烷基硫酸钠聚丙烯酰胺凝胶切片分析证实抑制活性存在于γ亚基中。纳摩尔浓度的γ亚基可阻断微摩尔浓度的Tα-GTPγS对PDE的激活。PDE的激活程度与γ亚基和Tα-GTPγS的浓度呈反比。在转导素激活PDE的过程中，γ亚基始终与盘状膜结合。γ亚基与天然PDE的αβ亚基结合非常紧密；解离常数小于10皮摩尔，这表明在没有Tα-GTP的情况下，视杆细胞外段中每1700个PDE分子中被激活的分子不到1个。
